Conditioned Media from Head and Neck Cancer Cell Lines and Serum Samples from Head and Neck Cancer Patients Drive Catabolic Pathways in Cultured Muscle Cells

Abstract

Background: The role of secreted factors from the tumor cells in driving cancer cachexia and especially muscle loss is unknown. We wanted to study both the action of secreted factors from head and neck cancer (HNC) cell lines and circulating factors in HNC patients on skeletal muscle protein catabolism.

Methods: Conditioned media (CM) made from head and neck cancer cell lines and mix of sera from head and neck cancer (HNC) patients were incubated for 48 h with human myotubes. The atrophy and the catabolic pathway were monitored in myotubes. The patients were classified regarding their skeletal muscle loss observed at the outset of management.

Results: Tumor CM (TCM) was able to produce atrophy on myotubes as compared with control CM (CCM). However, a mix of sera from HNC patients was not able to produce atrophy in myotubes. Despite this discrepancy on atrophy, we observed a similar regulation of the catabolic pathways by the tumor-conditioned media and mix of sera from cancer patients. The catabolic response after incubation with the mix of sera seemed to depend on the muscle loss seen in patients.

Conclusion: This study found evidence that the atrophy observed in HNC patients cannot be solely explained by a deficit in food intake.

Keywords: conditioned media; head and neck cancer; muscle cells protein metabolism; patient serum.

Figure 1

Effect of tumor cell-conditioned media or control-conditioned media on size and myosin content of C2C12-differentiated myotubes. (A) Picture of differentiated myotubes after incubation for 48 h with control-conditioned media (CCM). (B) Picture of differentiated myotubes after incubation for 48 h with tumor-conditioned media (TCM). (C) Average diameter analysis by microscopy of myotubes incubated with either CCM or TCM for 48 h (n = 231 for CCM and n = 324 for TCM), ****: p < 0.0001. (D) Number of myotubes classified according to their average diameter assessed by microscopy (in µm) stratified by media (CCM or TCM); <15: average myotube diameter <15 µm, [10,11,12,13,14,15,16,17,18,19,20]: average myotube diameter between 15 µm and 20 µm, [20,21,22,23,24,25]: average myotube diameter between 20 µm and 25 µm, ≥25: average myotube diameter ≥ 25µm. (E) Mean ± SD of myosin expression (heavy chain) assessed by Western blot analysis in C2C12 myotubes incubated with either TCM (n = 3) or CCM (n = 3).*: p < 0.05. AU: absorbance unit. The uncropped blots and molecular weight markers are shown in Supplementary Figure S1.

Figure 2

Myosin expression (heavy chain) in human primary myotubes incubated with either tumor conditioned media or control conditioned media. Mean ± SD of (heavy-chain) myosin expression levels in myotubes incubated with either tumor-conditioned media (TCM) (n = 3) or control-conditioned media (CCM) (n = 3, two tumor cell lines). ***: p < 0.001. AU: absorbance unit. The uncropped blots and molecular weight markers are shown in Supplementary Figure S2.

Figure 3

Change in mRNA expression in human primary myotubes incubated with either control-conditioned media (CCM) (n = 3 replicates) or with tumor-conditioned media (TCM) (n = 3 replicates). (A–C) Change in mRNA expression in human differentiated myotubes incubated for 48 h with either CCM or TCM. Gene expression in each condition was normalized to GAPDH expression. *: p < 0.05, **: p < 0.01, and ***: p < 0.001. (D): Time–course of proteasome activity in human-differentiated myotubes incubated with either CCM or TCM for different time-points (6 h, 24 h, and 48 h). ns: non significant.

Figure 4

(A) Autophagy in human primary myotubes incubated with either control-conditioned media (CCM) or with tumor-conditioned media (TCM). TCM: Tumor-conditioned media made from UT-SCC-5 and UT-SCC-60A cancer cell lines. CCM: control-conditioned media. CQ: chloroquine. For experiments with conditioned media, n = 3 replicates. *: p < 0.05. Level of the autophagy flux was defined at a level of 100 arbitrary unit in one replicate to enable comparison between experiments. The uncropped blots and molecular weight markers are shown in Supplementary Figure S3. (B) Illustration of a western blot used for the quantification of the autophagy flux shown in Figure 4A.

Figure 5

Myosin expression (heavy chain) in human primary myotubes incubated with either a mix of sera from cancer patients with mild or severe sarcopenia or from control participants. Mean ± SD of myosin expression (heavy chain) in myotubes incubated with either a mix of sera from cancer patients with severe sarcopenia (SS group), a mix of sera from cancer patients with mild sarcopenia (MS group), or a mix of sera from the control group. There were no significant differences found. A.U.: absorbance unit. For each condition, n = 2 replicates. The uncropped blots and molecular weight markers are shown in Supplementary Figure S4.

Figure 6

mRNA expression of human primary myotubes incubated for 48 h with a mix of sera from either control patients (control), HNC patients with severe sarcopenia (SS group), or HNC patients with mild sarcopenia (MS). For each condition, n = 2 replicates. Fold change in mRNA expression of human-differentiated myotubes incubated for 48 h with a mix of sera from either control patients (control), HNC patients with severe sarcopenia (SS group) or HNC patients with mild sarcopenia (MS). Gene expression in each condition was normalized to GAPDH expression. *: p < 0.05, **: p < 0.01, and ***: p < 0.001. (A) Genes related to the autophagy process. (B) Genes related to proteasomal activity. (C) Genes related to lipid metabolism, endoplasmic reticulum, or inflammatory processes.

Figure 7

Autophagy and proteasome activity in human primary myotubes treated with HNC-patient tumor-conditioned media. (A): Proteasome activity human-differentiated myotubes incubated for 48 h with a mix of sera from either control patients (control), HNC patients with severe sarcopenia (SS group), or HNC patients with mild sarcopenia (MS). (B): Autophagy flux in differentiated myotubes incubated with mix of sera from control or cancer patients (MS and SS groups). The uncropped blots and molecular weight markers are shown in Supplementary Figure S5. (C) Illustration of a blot used in this study for the experiment shown in B. For experiments made with mixed sera from HNC patients, n = 2 replicates. ** p < 0.01, ***: p < 0.001.